Our proposed research will be focused on two objectives: The first is to fill existing gaps in the immunogenetic analysis of the HLA-D system and to reach a more detailed knowledge of the polymorphism of DR, DQ and DR subregions. This study will be done at the protein level by the immunochemical approach we have developed. The second is to investigate the role that different Class II products play in the generation of HLA-associated diseases. By radioimmunoassay typing, several newly discovered markers, located on different Class II molecules, will be tested for their association with coeliac disease, insulin-dependent diabetes mellitus and rheumatoid arthritis. For the coeliac disease, an analysis at the DNA level will be performed with he aim of defining the precise disease-associated sequence site. Specifically, we will direct our effort toward the following projects: 1) Analysis of HLA Class II molecules controlled by different DR haplotypes and of those expressed on mouse cells transfected with a single DR Beta gene by immunospecifically isolating the Class II molecules and analyzing their structural features by two-dimensional peptide mapping. 2) Subunit assignment of DQ alloantigenic determinants by serologically analyzing the Class II molecules expressed on mouse cells transfected either with DQw2 Alpha and DQw3 Beta genes or with DQw2 Beta and DQw3 Alpha genes. 3) Serological detection of HLA-DP allospecificity by isolating DP molecules from mouse cells transfected with DP Alpha and Beta genes and testing their reactivity with anti-Class II alloantisera by the direct binding assay. 4) HLA-disease association studies by typing patients with coeliac disease, insulin-dependent diabetes mellitus and rheumatoid arthritis for newly found "haplotype-discriminating specificities". 5) Molecular genetic study of coeliac disease by sequencing the Beta domain of DQw2 molecules expressed on cells of B-cell lines established from DR3-homozygous coeliac disease patients by using the primer extension method.